7-hydroxyepiandrosterone having neuroprotective activity

ABSTRACT

7-Hydroxyepiandrosterone may be used for protection against acute or chronic neuronal damage.

This application is a 371 of PCT/GB01/2937 filed Jun. 29, 2001

The present invention relates to the use of certain 7-hydroxy-steroidcompounds for protection against neuronal cell death, and which are thususeful in the treatment and prevention of such conditions or thesequelae of such conditions as Alzheimer's Disease, Parkinson's Disease,Cognitive Impairment No Dementia (CIND), stroke, brain trauma, spinalcord injury and peripheral nerve injury, it is also useful for enhancingcognitive function.

The production of 7α-hydroxylated metabolites of dehydroepiandrosterone(DHEA) in vivo has been known since 1959 with the identification of7α-hydroxy-DHEA in urine [J J Schneider, M L Lewbart, Recent Progr.Horm. Res. 15 (1959) 201-230; L Starka et al, Clin. Chim. Acta 7 (1961)309-316)]. Since then, extensive 7α-hydroxylation of 3β-hydroxysteroidsubstrates (including DHEA and epiandrosterone—EPIA) has been reportedin tissue preparations from many human organs, including adult andfoetal liver, testes, epididymus, skin, mammary tissue, prostate,adipose stromal cells and tonsils. Hydroxylation of DHEA at the7-position has also been demonstrated in rat liver and in numerous mousetissues and organs. In all these studies, 7α-hydroxy-DHEA was by far themajor metabolite produced. Indeed, Doostzadeh et al [Steroids 63 (1998)608-614] reported that the production rate of 7α-hydroxy-DHEA by mouseliver microsomes was more than fifteen times the production rate of7β-hydroxy-DHEA.

EPIA, DHEA and pregnenolone have also been shown to be rapidly andextensively transformed to their corresponding 7α-hydroxy metabolites inthe rat brain [J M Guiraud et al, Steroids 34 (1979) 241-248; M Warneret al, Endocrinology 124 (1989) 2699-2706; Y Akwa et al, Biochem. J. 288(1992) 959-964)].

WO97/37664 discloses the use of certain specific compounds, including7α-hydroxy-substituted steroids, to treat neuropsychiatric, immune orendocrine disorders. Among the disorders suggested in WO97/37664 thatthese compounds may be used to treat is included Alzheimer's Disease.However, the mechanism suggested for this action is that the disorder ishypothesised to result from a deficit of the 7α-hydroxy-substitutedsteroid in the brain, and the treatment proposed in WO97/37664 thusrectifies this deficit by the administration of a 7α-hydroxy-substitutedsteroid to replace the missing compound. The procedure described inWO97/37664 thus treats an existing condition, rather than preventing thecondition or preventing a worsening of the condition by preventingfurther neuronal damage. WO97/37664 does not, therefore, describe aneuroprotective effect. It also does not suggest that the compounds maybe used to prevent the damage caused by sudden and traumatic events suchas stroke.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show graphs representing the number of morphologicalintact hippocampal CA1 pyramidal cells 7 days after global cerebralischemia in rats and under the influence of different compounds.

FIG. 1A, the data are presented as mean number±SEM of intact neurons per400 μm length of CA1 region.

FIG. 1B, the data are expressed as percentage of intact neurons per 400μm length of CA1 region compared to sham operated animals set as 100%.

FIG. 1C, the data are presented as absolute percentage ofneuroprotection when the number of surviving neurons in the ischemiagroup was set to zero and those of the sham operated group was set to100%.

We have now surprisingly discovered that 7-hydroxy-epiandrosterone,whether α, β or a mixture, can be used to protect against acute andchronic neuronal damage caused by such events as stroke, brain traumaand cerebral ischaemia such as may be induced by sub-arachnoidhaemhorrage or which occurs during heart bypass surgery etc.

In events such as prolonged hypoxia and ischaemia, which may or may notbe associated with hypoglycaemia, neuronal damage, to varying degrees,is encountered.

Ischaemia typically occurs during heart attacks, but the damage incurredat these times is substantially limited to the heart tissues, andcertain treatments have been developed. With regard to the presentinvention, we are concerned with the effects of more long term ischaemiaon the brain, such as occurs with stroke patients or as a result of headinjury. The severity of the ischaemia depends on the nature of thestroke or injury, but, invariably, there is brain damage, and it is thiswhich the present invention addresses.

Various neuroprotective agents are known in the art which attempt toalleviate the problem of brain damage, but all of those currently knowntend to be associated with adverse side effects. For example, MK801(dizocilpine maleate) is a fairly simple molecule and is known toprovide a level of neuroprotection to ischaemic patients. However, MK801is also associated with “alarming psychotropic effects” (Martindale), aswell as adverse motor effects. The neuroprotective effects are detailedin Brain Research 755 (1997) 3646 (Pringle, A. K., et al), incorporatedherein by reference. These same authors also described theneuroprotective effects of conotoxin in an earlier paper but, despitethe neuroprotective effects of this compound, adverse side effects, invivo, are observed.

Thus, the present invention consists in the use for the manufacture of amedicament for protection against acute or chronic neuronal damage of7-hydroxyepiandrosterone (7-hydroxy-EPIA).

This compound may be represented by the formula (I):

and the 7α and 7β isomers are, respectively:

The invention also embraces the use of precursors of these compounds andcompounds which, in vivo, are metabolised to give these compounds.

The α and β isomers may be used alone or in admixture, and, if inadmixture, may be present in any proportions. However, the 7β-isomerappears to show greater activity and is therefore presently preferred.

The compounds of the present invention may be prepared by a variety ofprocesses, well known in themselves, starting from the parent steroids.For example, they may be prepared by the methods described in theliterature referred to above, which would give a mixture of the 7β andcorresponding 7α compounds, which may then be separated by well knowntechniques.

As an example, 7α and/or 7β-hydroxy EPIA may be obtained from DHEA byallylic oxidation after protection of the 3β-hydroxy group and the17-ketone group using conventional methods. The product is then reducedwith a soluble metal compound catalyst (such as sodium hydride) and the3β-hydroxy and 17-ketone groups are deprotected. The 7α-hydroxy and7β-hydroxy epimers may then, if desired, be separated by conventionalmeans, for example column chromatography, and the 7α- and 7β-hydroxyEPIA may be crystallised to purity.

Alternatively, 7α- and 7β-hydroxy-EPIA may be prepared as illustrated bythe following reaction scheme:

In this reaction scheme, DHEA (II) is acetylated to give thecorresponding acetate of formula (III), which is then reacted withethylene glycol, to give the ketal of formula (IV). The ketal (IV) isthen oxidised as described in Example 3, to give the corresponding7-keto compound (V), which is then deacetylated, to give the compound offormula (VI). This is reduced, to give 7-hydroxy-17-ketal-EPIA offormula (VII), which is then treated with an acid to remove the ketalgroup and give 7-hydroxy-EPIA, which is finally separated into the 7β-and 7α-isomers by chromatography, to give 7α-hydroxy-EPIA (In and7β-hydroxy-EPIA (X).

The compounds of the present invention may be applier to the patient ifit is suspected that they are in danger of an ischaemic event especiallya stroke or head injury, or if they are suspected of developing achronic neurodegenerative disease, such as Alzheimer's disease or CIND,which may be facilitated by chronic sub-threshold brain ischaemia or byreduced neuronal energy production, such as is frequently observed inthe ageing brain. Such prophylactic application may be exceedinglyuseful. However, it has also been demonstrated that the compounds of thepresent invention have useful activity, even if applied after anischaemic event, but it will be appreciated that it is preferred toadminister the compounds as soon as possible, in order to avoid as muchneuronal degeneration as possible. In some circumstances it may bedesirable to administer repeated doses, especially where the patientremains in danger of an ischaemic event.

Suitable methods of administration are generally by injection, in orderto achieve the desired result as soon as possible. Thus, intravenousinjection is particularly preferred but, in some circumstances it may bepreferable to administer the compound directly into the cerebrospinalfluid.

The dose of the compound of the present invention will vary dependingupon many factors, including the age, body weight and general conditionof the patient, as well as the mode, frequency and route ofadministration. However, a dose of from 0.01 to 50 mg/kg body weight isgenerally recommended, a dose of from 0.05 to 20 mg/kg body weight beingmore preferred. This may be administered in a single dose or in divideddoses.

The invention is further illustrated by the following non-limitingExamples, of which Examples 1 to 7 illustrate the preparation ofcompounds of the present invention and Examples 8 and 9 illustrate theiractivity. In Examples 1 to 7, the Roman numerals refer to the formulaein the reaction schemes shown above.

EXAMPLE 1 DHEA-3-acetate (III)

A solution of 50 ml of pyridine and 50 ml of acetic anhydride containing10 g of DHEA (II) (34.72 mmol) was heated to reflux for 4 hours. Thereaction medium was cooled, poured into water and extracted with ethylacetate. The organic phase was dried over anhydrous sodium sulphate andevaporated to dryness. 11.0 g of DHEA-3-acetate (m) (33.33 mmol, 96%),which was recrystallised from ethanol, were obtained.

EXAMPLE 2 17-Ketal-DHEA-3-acetate (IV)

A solution of 100 ml of toluene containing 5 g of DHEA-3-acetate (III)(15.15 mmol), 5 ml of ethylene glycol and a catalytic amount ofp-toluenesulphonic acid was heated to reflux with steam distillationusing a Dean-Stark apparatus for 24 hours. The reaction medium waspoured into 100 ml of a 10% w/v aqueous potassium carbonate solution.The organic phase was decanted. The aqueous phase was extracted withethyl acetate. The organic phases were combined and evaporated todryness. 5.10 g of 17-ketal-3-DIEA-acetate (IV) (13.64 mmol, 90%), whichwas recrystallised from ethanol, were obtained.

EXAMPLE 3 7-Keto-17-ketal-DHEA-3-acetate (V)

A solution of 70 ml of pyridine containing 5 g of17-ketal-DHEA-3-acetate (IV) (13.37 mmol) and a catalytic amount ofBengal Rose was irradiated using a medium-pressure mercury vapour lampwith oxygen sparging. A catalytic amount of copper acetate was added tothe reaction medium after 24 hours. The reaction medium, after 24 hours,was evaporated to dryness. The residue was purified by flashchromatography (SiO2/ethyl acetate:cyclohexane 3/7). 3.11 g of7-keto-17-ketal-DHEA-3-acetate (V) (8.02 mmol, 60%) were obtained.

EXAMPLE 4 7-Keto-17-ketal-DHEA (VI)

A solution of 50 ml of methanol containing 1% of potassium hydroxide and1 g of 7-keto-17-ketal-DHEA-3-acetate (V) (2.58 mmol) was heated toreflux for 2 hours. The reaction medium was then cooled, neutralised andthen extracted with ethyl acetate. The organic phase was dried overanhydrous sodium sulphate and then evaporated to dryness. 802 mg of7-keto-17-ketal-DHEA 5 (2.32 mmol, 90%), which was recrystallised frommethanol, were obtained.

EXAMPLE 5 7-Hydroxy-17-ketal-EPIA (VII)

10 g of 7-keto-17-ketal-DHEA (VD (28.90 mmol) were added to a liquidammonia solution at −33° C. containing 2.65 g of sodium. After 4 hours,ammonium chloride was added until the blue colour disappeared. 2.65 g ofsodium were then added. After 4 hours, ammonium chloride was again addeduntil the blue colour disappeared. Water was added and the ammonia wasallowed to evaporate. The reaction medium was extracted with ethylacetate. The organic phase was dried over anhydrous sodium sulphate andthen evaporated to dryness. 6.07 g of 7-hydroxy-17-ketal-EPIA CM (17.34mmol, 60%) were obtained.

EXAMPLE 6 7-Hydroxy EPIA (VIII)

A solution of 100 ml of acetone containing 5 ml of water, 10 g of7-hydroxy-17-ketal-EPIA (VII) (28.57 mmol, 50%) and a catalytic amountof p-toluenesulphonic acid was heated to reflux for 4 hours. Thereaction medium was cooled, poured into 100 ml of a 10% w/v aqueoussodium carbonate solution and then extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulphate and thenevaporated to dryness. The residue was purified by flash chromatography(SiO₂/ethyl acetate). 5.24 g of 7-hydroxy-EPIA (VIII) (17.14 mmol, 60%)were obtained.

EXAMPLE 7 7α-Hydroxy-EPIA (IX) & 7β-hydroxy-EPIA (X)

7-Hydroxy-EPIA (VIII) (5 g) containing 7α and 7β epimers in a ratio65/35 was purified by flash chromatography (Al₂O₃/CHCl₃).7β-Hydroxy-EPIA (X) (2.5 g) was obtained first, before 7α-hydroxy-EPIA(IX) (1.34 g). 7β-Hydroxy-EPIA (X) and 7 cc-hydroxy-EPIA (IX) wererecrystallised from ethyl acetate.

EXAMPLE 8 Protocol for Studying Hypoxic Neuronal Damage

Organotypic hippocampal slice cultures were prepared using the basicmethod of Pringle et al (1996, 1997) modified as follows:

Wistar rat pups (8-11 days old) were decapitated and the hippocampusrapidly dissected into ice-cold Gey's balanced salt solutionsupplemented with 4.5 mg/ml glucose. Slices were separated and platedonto Millicell CM culture inserts (4 per well) and maintained at 37°C./5% CO₂ for 14 days. Maintenance medium consisted of 25%heat-inactivated horse serum, 25% Hank's balanced salt solution (HBSS)and 50% minimum essential medium with added Earle's salts (MEM)supplemented with 1 mM glutamine and 4.5 mg/ml glucose. Medium waschanged every 3-4 days.

Experimental hypoxia was performed as described previously (Pringle etal., 1996; 1997). Briefly, cultures were transferred to serum freemedium (SFM—75% MEM, 25% HBSS supplemented with 1 mM glutamine and 4.5mg/ml glucose) containing 5 μg/ml of the fluorescent exclusion dyepropidium iodide (PI). Cultures were allowed to equilibrate in SFM for60 minutes prior to imaging. PI fluorescence was detected using a Leicainverted microscope fitted with a rhodamine filter set. Any cultures inwhich PI fluorescence was detected at this stage were excluded fromfurther study. Hypoxia was induced by transferring cultures to SFM (+PI)which had been saturated with 95%N₂/5%CO₂. Culture plates (without lids)were then sealed into an airtight chamber in which the atmosphere wassaturated with 95%N₂/5%CO₂ by continuously blowing through gas at 10L/min for ten minutes before being sealed and placed in the incubatorfor 170 mins (total time of hypoxia was therefore 180 mins). At the endof the hypoxic period cultures were returned to normoxic SFM containingPI and placed back in the incubator for 24 hours.

Neuronal damage was assessed as described previously (Pringle et al.,1996; 1997) using either NIH Image 1.60 running on an Apple IIsicomputer or OpenLab 2.1 (Improvision) running on a Macintosh G4/400.Images were captured using a monochrome camera and saved onto opticaldisk for offline analysis. Light transmission images were captured priorto the addition of drugs, and PI fluorescence images recorded at the endof the 24-hour post-hypoxia recovery period. The area of the CA1 celllayer was determined from the transmission image. The area of PIfluorescence in CA1 was measured using the density slice function withinNIH image or Openlab, and neuronal damage expressed as the percentage ofthe CA1 in which PI fluorescence was detected above background.

Steroid compounds were prepared by making an initial 1 mg/ml solution inethanol and further diluting down in SFM. Compounds were added to thecultures for 45 minutes prior to hypoxia, during the hypoxic episode andduring the post-hypoxic recovery period. Control experiments consistedof cultures treated with vehicle alone.

Results

Experiment 1:

An initial experiment was performed to determine whether 7βOH-EPIA and7βOH-EPIA were neuroprotective at a high concentration of 100 nM.Hypoxia produced a lesion in 25.5±6.4% of CA1. This damage wassignificantly reduced by both 7αOH-EPIA and 7βOH-EPIA when present pre-,during and post-hypoxia as shown in Table I, below.

TABLE 1 Compound N % Damage in CA1 Control Hypoxia 17 25.5 ± 6.4 Hypoxia + 100 nM 7αOH-EPIA 16  4.0 ± 2.9** Hypoxia + 100 nM 7βOH-EPIA 16 9.0 ± 4.7* Experiment 2:

Having determined that both the α- and β-isomers of 7OH-EPIA wereneuroprotective, we assessed the concentration-dependency of thiseffect. Control hypoxia resulted in neuronal damage to 31.9±4.7% of theCA1.7αOH-EPIA was significantly protective at 100 nM. A small, butnot-statistically significant reduction, in neuronal damage was observedat 10 nM, and there was no effect at 1 nM. In contrast, 7βOH-EPIA wassignificantly neuroprotective at 10 nM and 100 nM, but activity was lostif the concentration was reduced to 1 nM. (See Table 2).

TABLE 2 Compound N % Damage in CA1 Control Hypoxia 29 31.9 ± 4.7Hypoxia + 1 nM 7αOH-EPIA 14 28.8 ± 5.8 Hypoxia + 10 nM 7αOH-EPIA 15 21.9± 8.1 Hypoxia + 100 nM 7αOH-EPIA 16  11.8 ± 2.8** Hypoxia + 1 nM7βOH-EPIA 15 20.6 ± 7.2 Hypoxia + 10 nM 7βOH-EPIA 12  11.9 ± 4.7*Hypoxia + 100 nM 7βOH-EPIA 13  14.3 ± 5.0*

EXAMPLE 9 Global Cerebral Ischemia in Rats (4 Vessel Occlusion)

Cerebral ischemia was induced by four-vessel-occlusion (4VO) in maleWistar rats (250-280 g). Both vertebral arteries were occluded byelectrocauterization in pentobarbital anesthesia (60 mg/kg i.p.). Theanimals were allowed to recover for 24 hours with free access to waterbut not food. The next day the carotid arteries were exposed under 2%halothane in 30% oxygen/70% nitrous oxide anesthesia and were occludedfor 10 minutes using microvascular claps. Subsequently, both clamps wereremoved and both arteries were inspected for immediate reperfusion.During the operation and the following 3 hours normothermia of theanimals (37.5Γ0.5° C.) was maintained by using a thermostaticallycontrolled heating blanket connected to a rectal thermometer. Forcontrol, in sham-operated animals both vertebral arteries werecauterized in pentobarbital anesthesia and both common carotid arterieswere exposed but not clamped under 2% halothane in 30% oxygen/70%nitrous oxide anesthesia the following day. The wound was treated withlidocaine gel and then sutured. The animals were kept under a heatinglamp at 30° C. environmental temperature until they regainedconsciousness.

Seven groups of animals were investigated:

-   -   1. (n=8) steroid compound, 7β-OH EPIA (0.1 mg/kg, i.v. via tail        vein, three injections: 15 minutes prior to the induction of        ischemia, during ischemia and 5 minutes after reperfusion);    -   2. (n=8) steroid compound, 7β-OH EPIA (0.3 mg/kg, i. v. three        injections as described in 1.);    -   3. (n=8) steroid compound, 7β-OH EPIA (1 mg/kg, i. v., three        injections as described in 1.);    -   4. (n=8) NBQX (disodium salt, because more water soluble) as        reference substance and positive control (TOCRIS, Germany, 30        mg/kg, i. p., three injections as described in 1.);    -   5. (n=8) received vehicle (0.9% NaCl, containing 100 μl Ethanol)        three injections as described in 1.);    -   6. (n=8) ischemia alone;    -   7. (n=8) sham operated controls.

NBQX is 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and isknown to have neuroprotective activity [Gill, R., Nordholm, L., LodgeD.: The neuroprotective action of2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) in a ratfocal ischaemia model. Brain Res. 580, 35-43, 1992].

7β-OH EPIA is 7β-hydroxyepiandrosterone, a compound of the presentinvention.

The substances were dissolved in 100 μl Ethanol and finally diluted with0.9% NaCl.

After a survival time of 7 days after ischemia, all animals wereperfusion fixed transcardially with 4% paraformaldehyde. The brains werethen removed carefully and postfixed in the same fixative for 2 hours.After cryoprotection in 30% sucrose, the brains were rapidly frozen inisopentane and stored at −80° C. Twenty-micrometer cryostat sectionscomprising the hippocampal formation were Nissl stained with toluidineblue or NeuroTrace fluorescence.

Data Analysis:

The severity of neuronal damage in the hippocampal CA1 region afterischemia was evaluated by the number of surviving neurons using Nisslstaining. The mean number of morphologically intact neurons per 400 μmlength was calculated in CA1 region for each group. Cell counting wasperformed in 3-5 serial sections per animal and 6 times 400 μm CA1 areaper section using a light microscope equipped with a 20× objective. Thedata were statistically analyzed by paired Student's t-test. Data werepresented as mean±SEM.

Results and Discussion

The results are shown in FIGS. 1A, 1B, and 1C of the accompanyingdrawings.

Morphological intact hippocampal CA1 neurons were characterized by Nisslstaining (toluidine blue and NeuroTrace) with the following criteria:clear shape of a neuronal perikarya, large nucleus with a positivelabeled nucleolus, a small cytoplasm zone around the nucleus withpositive Nissl staining, indicating the intact rough endoplasmicreticulum with ribosomes and therefore the intact protein synthesismachinery.

10 minutes of global ischemia (mild ischemia) and a survival time of 7days leads to a neurodegeneration of pyramidal cells selectively in thehippocampal CA1 region (FIGS. 1A-1C). The mean number of pyramidal cellsin CA1 of sham operated animals was 121.5Γ4.3 (set as 100%). Therefore,60% of CA1 neurons died after 10 minutes of global ischemia (FIG. 1B).The number of neurons in the animal group of ischemia and i. v.injection of vehicle (NaCl plus 100 μl Ethanol) applied as described inthe experiment was comparable to that of the ischemia group alone (FIGS.1A, 1B). NBQX (30 mg/kg, i.v., three injections as described in theexperiment) showed a significant (p=0.03) neuroprotection in CA1pyramidal cells compared to the ischemia group. Compared to the ischemiaalone NBQX leads to a 47.5% neuroprotection while compared to the shamoperated animals the protective effect was 68.5%. The neuroprotectioncaused by NBQX was in agreement with Gill et al., 1992 and Gill 1994demonstrating the validity of the global ischemia model we used in ourexperiments. 7β-OH EPIA leads to a concentration dependentneuroprotection of hippocampal CA1 pyramidal cells after 10 minutes ofglobal ischemia and a survival time of 7 days (FIG. 1A). T-test analysisrevealed a highly significant neuroprotective effect of 7β-OH EPIA inconcentrations of 0.1 mg/kg (p=0.01) and 0.3 mg/kg (p=0.0008). Comparedto the sham operated group 7β-OH EPIA showed a 74.8% (0.1 mg/kg) and a83.9% (0.3 mg/kg) neuroprotective effect on CA1 pyramidal cells,respectively (FIG. 1C). 7β-OH EPIA in a concentration of 1.0 mg/kgshowed only a tendency to neuroprotection, but the effect was notsignificant.

In all experiments with 7β-OH EPIA injected i.v. prior, during and afterischemia we never observed any behavioral abnormalities of the animals.

1. A method of treating neuronal damage in a mammal comprising administering thereto an effective amount of 7β-hydroxyepiandrosterone.
 2. The method according to claim 1, wherein the neuronal damage is caused by stroke or brain trauma.
 3. The method according to claim 1, wherein the neuronal damage is caused by Alzheimer's Disease, Parkinson's Disease, Cognitive Impairment No Dementia, spinal cord injury, or peripheral nerve injury.
 4. A method for reducing neuronal cell death following acute neuronal damage in a mammal comprising administering thereto an effective amount of 7β-hydroxyepiandrosterone.
 5. The method according to claim 4, wherein the acute neuronal damage is caused by stroke.
 6. The method according to claim 4, wherein the acute neuronal damage is caused by spinal cord injury.
 7. A method for reducing neuronal cell death following chronic neuronal damage in a mammal comprising administering thereto an effective amount of 7β-hydroxyepiandrosterone.
 8. The method according to claim 7, wherein the chronic neuronal damage is caused by Alzheimer's Disease.
 9. The method according to claim 7, wherein the chronic neuronal damage is caused by Parkinson's Disease. 